1. Field of the Invention
The present invention relates to a method of inactivating cytosolic aspartate aminotransferase isozyme comprising addition of a specific inhibitory enzyme, to a method for the fractional determinations of aspartate aminotransferase (EC 2.6.1.1, systematic name=L-Aspartate: 2-oxoglutarate aminotransferase; another name=Glutamic-oxaloacetic transaminase; hereinafter referred to as AST) isozymes present in specimens of blood serum, blood plasma, etc., and to a composition therefor. More particularly, the invention relates to a method for the practional determination of two AST isozymes, which comprises (a) inactivating the cytosolic AST isozyme thereof with an enzyme which specifically inactivates this isozyme, followed by determining the residual isozyme activity, i.e. the mitochondrial AST isozyme according to a known method for AST activity determination; and (b) calculating the activity of the cytosolic AST by subtracting the activity of mitochondrial AST determined in the above-mentioned (a) from the total activity of AST isozymes which has been estimated according to a known method.
2. Description of the Prior Art
AST isozymes are known as enzymes occurring in the liver, myocardium, brain, skeletal muscle, kidney and the like, and catalyzing the following reaction:
L-Aspartate+2-Oxoglutarate PA1 .revreaction.Oxaloacetate+L-Glutamate
The AST isozymes include two kinds of isozymes different in localization, one being cytosolic AST (hereinafter referred to as "s-AST") and the other being mitochondrial AST (hereinafter referred to as "m-AST"). The fractional determination thereof is useful for the clinical diagnosis of hepatitis, myocardial infraction, etc.
Up to now the assay of AST isozymes has been performed according to a chromatographic method using an anionic ion exchanger (Rinsho Kagaku, 5, 163, (1977)), an imnunochemical method, or an electrophoretic method. However, these methods have various disadvantages such that the procedure is complicated and time-consuming and the sensitivity and accuracy of estimation are insufficient.
On the susceptibility of AST isozymes to proteases, there are reports by E. Sandmeier et al. (J. Biol. Chem., 255, 10284-10289 (1980)) and by D E. Metzler et al. (Federation Proceedings, 41, 2432-2436 (1982)). E. Sandmeier et al. have reported that trypsin limitedly cleaved m-AST to inactive it, and in a preliminary experiment, a similar proteolytic cleavage of s-AST was observed. D. E. Metzler et al. have described that m-AST was inactivated by trypsin though more slowly than s-AST.